Central nervous system (CNS) disorders are major public health issues. For example, Parkinson's disease (PD), the second most common neurodegenerative disease in the United States, affects over 1 million people while imposing an annual cost to the U.S. economy of 5.9 billion dollars. Clinically, PD is characterized by, among other signs, a decrease in spontaneous movements, gait difficulty, postural instability, rigidity, and tremor; these clinical signs are a direct result of the degeneration of the pigmented neurons (i.e., dopaminergic neurons) in the substantia nigra of the basal ganglia region of the brain. The progressive degeneration of the substantia nigra leads to decreased availability of dopamine, as the pigmented neurons of the substantia nigra are the sites of synthesis of this important catecholamine neurotransmitter.
Dopamine is synthesized in the terminal nerve endings of the dopaminergic neurons of the substantia nigra, specifically the substantia nigra pars compacta. The dopaminergic nerves project into the corpus striatum, specifically innervating the putamen and the caudate nucleus. Three enzymes are necessary for the efficient biosynthesis of dopamine: tyrosine hydroxylase (TH), guanosine triphosphate cyclohydrolase I (GCH), and aromatic L-amino acid decarboxylase (AADC). Tyrosine hydroxylase adds a hydroxyl group to the amino acid tyrosine creating L-dihydroxyphenylalanine (L-dopa). The enzymatic activity of TH requires the necessary cofactor tetrahydrobiopterin (BH4), GCH catalyzing the first and rate-limiting step of the biosynthesis of BH4. Lastly, AADC removes the terminal carboxyl group of L-dopa, which results in the formation of dopamine.
Currently, many CNS disorders such as PD are treated by systemic administration of a therapeutic agent. Systemic administration, however, can be ineffective because of a drug's inability to pass through the blood-brain-barrier and/or because of the potential for deleterious side effects. Thus, many potentially useful compounds, such as proteins, cannot be administered systemically. Treatment of PD currently involves oral administration of L-dopa, often in combination with a peripheral inhibitor of AADC. As PD progresses, a majority of patients experience a reduction in AADC content in affected regions of the brain (i.e., the substantia nigra). Since AADC converts L-dopa to dopamine, escalating doses of L-dopa are required for therapeutic efficacy, but this often results in increased side effects. Moreover, as the substantia nigra progressively deteriorates, AADC depletion continues unabated, often reaching a level where therapeutic benefit derived from administration of L-dopa is no longer realized.
In view of the limitations of current systemic therapies, gene delivery is a promising method for the treatment for CNS disorders such as PD. A number of viral based systems for gene transfer purposes have been described, including systems based on retroviruses and adenoviruses.
Adeno-associated virus (AAV) systems are emerging as the leading candidates for use in gene therapy. AAV is a helper-dependent DNA parvovirus that requires infection with an unrelated virus such as adenovirus, a herpesvirus or vaccinia, in order for a productive infection to occur. The helper virus supplies accessory functions that are necessary for most steps in AAV replication.
AAV infects a broad range of organisms as well as tissue types, without eliciting the cytotoxic effects and adverse immune reactions in animal models that have been observed with other viral vectors (see, e.g., Muzyczka, (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Flotte et al. (1993) Proc. Natl. Acad. Sci. USA 90:10613-10617; Kasseiser et al. (1992) Gene Therapy 1:395-402; Yange et al. Proc. Natl. Acad. Sci. USA 91:4407-4411; Conrad et al. (1996) Gene Therapy 3:658-668; Yang et al. (1996) Gene Therapy 3:137-144; Brynes et al. (1996) J. Neurosci. 16:3045-3055). Because it can transduce non-dividing tissue, AAV may be well adapted for delivering genes to the central nervous system (CNS); indeed, AAV vectors containing therapeutic genes have been shown to transduce mammalian brain (see, e.g., During et al., (1998) Gene Therapy 5:820-827; Mandel et al. (1998) J. Neurosci. 18:4271-4284).
AAV vector delivery of dopamine-synthesizing enzymes to mammalian brain tissue has been described. Using a single gene approach, Kaplitt et al. (1994) Nature Genetics 8:148-153) stereotaxically injected recombinant AAV virions containing the gene coding for human tyrosine hydroxylase (rAAV-TH) to the denervated striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats. Both neurons and glial cells were transduced. Delivery of rAAV-TH to monkey striatum resulted in expression of TH for up to 2.5 months (During et al., supra). Bankiewicz et al. demonstrated therapeutic levels of dopamine synthesis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys after transduction of striatal cells with rAAV-AADC followed by oral administration of L-Dopa (Bankiewicz et al. (2000) Exp Neurol 164:2-14). Sanchez-Pemaute et al. demonstrated behavioral improvements in 6-OHDA-lesioned rats after transduction of striatal cells with rAAV-AADC (Sanchez-Pemaute et al. (2001) Mol Ther 4:324-330).
In a dual gene approach, Mandel et al., supra, co-transduced 6-OHDA-lesioned rats with AAV virions containing either TH or GCH genes (rAAV-TH and rAAV-GCH). Standard methods detected the presence of TH and GCH in the striatum of the 6-OHDA-lesioned rats. Similarly, in experiments described in During et al., supra, MPTP-treated green monkeys were co-transduced with rAAV-TH and rAAV virions containing the human AADC gene (rAAV-AADC). Gene expression was verified using standard techniques. Similarly, Fan et al. transduced 6-OHDA-lesioned rats after co-transduction of the striatum with rAAV-TH and rAAV-AADC virions (Fan et al. (1998) Hum Gene Ther. 9:2527-2535). Striatal levels of TH and AADC were measured using immunohistochemical staining. Rats expressing TH and AADC demonstrated increased behavioral recovery from 6-OHDA lesions than rats expressing TH or AADC alone.
Gene therapy for the treatment of PD has focused on transducing one or two dopamine biosynthetic enzymes into the CNS of animal models of PD. Under normal physiological conditions, efficient dopamine synthesis requires the presence of all three enzymes (Elsworth et al. (1997) Exp. Neurol. 144:4-9), which are anterogradely transported along the dopaminergic projections of the substantia nigra in the direction of the corpus striatum (for a detailed discussion see Nagatsu et al. (1990) Basic, Clinical, and Therapeutic Aspects of Alzheimer's and Parkinson's Disease. T. Nagatsu, A. Fisher, and M. Yoshida, eds. (Plenum Press, New York) pp. 263-266).
As discussed above, the dopaminergic neurons of the substantia nigra are depleted over time in patients with PD, greatly reducing dopamine synthesis and delivery to the corpus striatum; such a reduction culminates in the severe effects of advanced disease. By providing methods for the delivery and expression of all three enzymes in the corpus striatum of patients with PD, increased dopamine synthesis and hence increased therapeutic efficacy may be obtained. Such methods are disclosed herein.